The long term objectives of our studies are to define the structural and functional domains of the sodium-potassium ATPase (Na+K+ATPase), and to describe the number, sequence organization and regulation of the genes which code for it. The Na+K+ATPase is an integral membrane protein which directly couples the hydrolysis of ATP with the vectorial transport of sodium and potassium ions across the plasma membrane. The enzyme consists of a large catalytic subunit, Alpha, with a molecular weight of 110,000 and a smaller glycoprotein subunit, Beta, of unknown function, with a molecular weight of about 55,000. A third subunit, Gamma may also exist. Not only is the Na+K+ATPase an essential mammalian cell enzyme but it also appears to be the pharmacological receptor for cardiac glycosides. Thus detailed information concerning the structural and functional domains of this enzyme is important not only for understanding ion transport but also for defining the interaction of the Na+K+ATPase with cardiac glycosides. During the last grant period we constructed and isolated a cDNA clone which contains the entire coding region of the catalytic subunit of the sheep kidney enzyme. The entire nucleotide sequence of the clone has been determined, the complete amino acid sequence of the Alpha subunit deduced, and a number of structural and functional domains identified. The specific aims for the current proposal represents an extension of these studies. We will clone the gene or genes for the Alpha subunit of the sheep Na+K+ATPase and determine their sequence organization. We will also determine the structural basis for the multiple forms of the Alpha subunit. Several lines of evidence demonstrate that at least two Alpha subunits designated Alpha and Alpha(+) exist in some tissues including brain and adipocytes and that this difference resides in the primary structure of the subunits. This question will be resolved by preparing cDNA libraries of tissues having Alpha and Alpha(+) subunits and characterizing these cDNAs by restriction endonuclease mapping and DNA sequence analysis. After identifying the structural difference between the cDNAs the genetic basis of the difference will be examined. Finally we will determine if an altered Alpha subunit is the basis for ouabain resistance. Other laboratories including our own have isolated cell lines whose growth is resistant to ouabain and it is hypothesized that resistance involves the Alpha subunit. The genes for the Alpha subunit will be cloned from resistant cells and recombinants carrying the gene conferring ouabain resistance will be identified by DNA mediated transfection and characterized. This study should determine whether the basis for resistance to this drug resides in the Alpha subunit and define the differences between the mutant and wild type genes.